Sequential Activation of Classic PKC and Estrogen Receptor α Is Involved in Estradiol 17ß-D-Glucuronide-Induced Cholestasis

Abstract

Estradiol 17ß-d-glucuronide (E17G) induces acute cholestasis in rat with endocytic internalization of the canalicular transporters bile salt export pump (Abcb11) and multidrug resistance-associated protein 2 (Abcc2). Classical protein kinase C (cPKC) and PI3K pathways play complementary roles in E17G cholestasis. Since non-conjugated estradiol is capable of activating these pathways via estrogen receptor alpha (ERα), we assessed the participation of this receptor in the cholestatic manifestations of estradiol glucuronidated-metabolite E17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHC). In both models, E17G activated ERα. In PRL, E17G maximally decreased bile flow, and the excretions of dinitrophenyl-glutathione, and taurocholate (Abcc2 and Abcb11 substrates, respectively) by 60% approximately; preadministration of ICI 182,780 (ICI, ERα inhibitor) almost totally prevented these decreases. In IRHC, E17G decreased the canalicular vacuolar accumulation of cholyl-glycylamido-fluorescein (Abcb11 substrate) with an IC50 of 91±1 µM. ICI increased the IC50 to 184±1 µM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Abcc2 substrate, glutathione-methylfluorescein. ICI also completely prevented E17G-induced delocalization of Abcb11 and Abcc2 from the canalicular membrane, both in PRL and IRHC. The role of ERα in canalicular transporter internalization induced by E17G was confirmed in ERα-knocked-down hepatocytes cultured in collagen sandwich. In IRHC, the protection of ICI was additive to that produced by PI3K inhibitor wortmannin but not with that produced by cPKC inhibitor Gö6976, suggesting that ERα shared the signaling pathway of cPKC but not that of PI3K. Further analysis of ERα and cPKC activations induced by E17G, demonstrated that ICI did not affect cPKC activation whereas Gö6976 prevented that of ERα, indicating that cPKC activation precedes that of ERα. Conclusion: ERα is involved in the biliary secretory failure induced by E17G and its activation follows that of cPKC.

EGFR participates downstream of ERα in estradiol-17β-d-glucuronide-induced impairment of Abcc2 function in isolated rat hepatocyte couplets

“…Our group has recently demonstrated that GPR30, a GPCR, and ERα accounts in part for the acute cholestasis caused by E17G Barosso et al 2012), event that correlates well with its ability to induce endocytic internalization of the canalicular transporters such as Abcc2 (Mottino et al 2003(Mottino et al , 2005. The actions of GPR30 and ERα in E17G-induced cholestasis constitute two independent pathways.…”

“…Based on the IC50 value, the concentration of E17G used for transport studies was 100 µM. This value is the estimated concentration at which hepatocytes are exposed in perfused rat livers (3 µmol per liver administered during a minute in a perfusing flow of 30 mL/ min, Barosso et al 2012).…”

An updated review on drug-induced cholestasis: Mechanisms and investigation of physicochemical properties and pharmacokinetic parameters

“…Furthermore, E 2 17G activates the estrogen receptor α (Erα) in isolated perfused rat liver 101. This might explain why chemical inhibition or knock‐down of ERα partially prevented decreased Bsep/Mrp2 activity and reduced transporter internalization 101. This finding is substantiated further by the observation that Erα(−/−) mice are resistant to ethinylestradiol‐induced hepatotoxicity 102.…”

“…E 2 17G activates classical, Ca 2+ ‐dependent protein kinase C and phosphoinositol 3‐kinase signaling pathways, which are cooperatively involved in internalization and intracellular retention of Bsep/Mrp2 99,100. Furthermore, E 2 17G activates the estrogen receptor α (Erα) in isolated perfused rat liver 101. This might explain why chemical inhibition or knock‐down of ERα partially prevented decreased Bsep/Mrp2 activity and reduced transporter internalization 101.…”

“…At the moment, it is not clear how activation of ERα leads to internalization of transporter proteins. Recent data suggest that phosphoinositol 3‐kinase is not directly involved in this process, and it was shown that activation of protein kinase C actually precedes activation of ERα, suggesting that other yet unknown mediators are involved 101. Interestingly, the upregulation of Mrp3 expression and activity in rat liver after administration of ethinylestradiol was independent of cholestasis and required the estrogen receptor 103…”